Humanized monoclonal antibodies and methods of use

ABSTRACT

Disclosed is a humanized monoclonal antibody that binds to the human immunoglobulin heavy chain variable region germline gene VHI-69. The antibody is derived from Mab G6 and recognizes the same epitope. Moreover, the antibody is used in combination with vaccines to augment an immune response to the antigen.

RELATED APPLICATIONS

This application is a national stage application, filed under 35 U.S.C.§371, of PCT Application No. PCT/US2011/038970, filed on Jun. 2, 2011,which claims the benefit of U.S. Ser. No. 61/350,790, filed on Jun. 2,2010, the contents of each of which are incorporated herein by referencein their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “20363-057N01US_ST25.txt”, which wascreated on Nov. 29, 2012 and is 16 KB in size, are hereby incorporatedby reference in their entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under grant number U01AI074518 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to humanized anti-humanVH1-69antibodies as well as to methods of using same to augment the immuneresponse to microbial infection.

BACKGROUND OF THE INVENTION

An influenza pandemic represents one of the greatest acute infectiousthreats to human health. The 1918-1919 influenza pandemic caused anestimated 500,000 deaths in the United States, making it the most fatalevent in all of US history. The spread of highly pathogenic avianinfluenza (HPAI) H5N1 influenza across Asia and now to the Middle Eastand northern Africa creates a substantial risk for a new pandemic toarise.

Natural variation as well as escape mutants suggests that continuedevolution of the virus should impact the decision on which strain(s)should be used for passive and active immunization. Although a number ofimportant epitope mapping and neutralization escape studies have beenreported new neutralizing antibodies and related structural studies areneeded to develop immunization strategies to develop a “universalvaccine” against a broad range of Group 1 influenza viruses. Thechallenges to developing a protective vaccine against Group 1 influenzaare formidable and new approaches are needed to prevent and treat humaninfection by an ever changing enemy. There is a need to rapidly developtherapeutic strategies to elicit protective host's immunity, bothpassively and actively.

SUMMARY OF THE INVENTION

The invention is based upon the discovery of monoclonal antibodies whichbind the human immunoglobulin heavy chain variable region germline geneVH1-69. The monoclonal antibody is fully human. In various aspects, themonoclonal antibody is a bivalent antibody, a monovalent antibody, asingle chain antibody or fragment thereof. Exemplary monoclonalantibodies include a monoclonal antibody that binds to the same epitopeas murine monoclonal antibody G6.

The monoclonal antibodies of the invention can have a binding affinitythat is 1 nM or less.

The monoclonal antibody has a heavy chain variable amino acid sequencecontaining SEQ ID NOS: 2 or 12, and/or a light chain variable amino acidsequence containing SEQ ID NO: 4. The monoclonal antibody has a heavychain variable nucleic acid sequence containing SEQ ID NOS: 1 or 11,and/or a light chain variable nucleic acid sequence containing SEQ IDNO: 3.

Also provided by the invention is a monoclonal human immunoglobulinheavy chain variable region germline gene VH1-69 antibody or fragmentthereof, where the antibody has a CDRH1: GYTFTSYW (SEQ ID NO: 5); CDRH2:VSPGNSDT (SEQ ID NO: 6); and CDRH3: TRSRYGNNALDY (SEQ ID NO: 7) and aCDRL1: QGISSNIVW (SEQ ID NO: 8); CDRL2: HGT (SEQ ID NO: 9); and CDRL3:VQYSQFPPT (SEQ ID NO: 10).

In some aspects the antibody according to the invention is covalentlylinked to an antigen. The antibody is preferably a single chain antibodyand the antigen is the stem region of the hemagglutinin (HA) protein ofan influenza virus.

Also included in the invention are methods of augmenting the immuneresponse of a subject to an antigen by administering to the subject ananti-immunoglobulin variable region germline gene idiotype antibody andan immunogen capable of inducing an immune reaction to the antigen. Insome aspects the antibody is covalently linked to the antigen. Thegermline gene encodes for a light chain polypeptide or a heavy chainpolypeptide. Preferably, the variable region germline gene is VH1-69.The antigen is a virus, a bacterium, or a fungus. For example the virusis an influenza virus. Preferably, the immunogen is the hemagglutinin(HA) protein of an influenza virus or fragment thereof. Most preferably,the immungen contains the stem region of the hemagglutinin (HA) proteinof an influenza virus. The antibody can be administered prior to,concurrently with, or subsequent to the administration of the immunogen.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows homology model of mouse G6 antibody heavy chain (VH) andlight chain (VL) generated using Web antibody modeling (WAM). VH coloredin blue and VL colored in green with CDRs highlighted in red.

FIG. 2 shows the comparison of antigen binding activity of huG6.1 andmG6 antibodies.

FIG. 3 shows the homology model of Human G6.1 antibody VH and VL. a),Human G6.1 antibody model, VH in blue and VL in green with CDRs in red.b), Human G6.1 homology model after GROMOS force field energyminimization, visualized in DeepView program, highlighted in differentcolors are residues exhibiting either distorted geometry or stericclashes.

FIG. 4 displays residues with distorted geometry and steric clashesbetween residues within framework regions as well as between frameworkand CDR residues using Pymol program.

FIG. 5 shows the comparison of antigen binding activity of huG6.2 andmG6 scFv-Fc.

FIG. 6 is a Real time Bio-Layer Interferometry binding analysis ofHuG6.2(Red) and Mouse G6(Blue) to the biotinylated D80 scFv-Fc.

FIG. 7 is a Real time Bio-Layer Interferometry binding analysis ofHuG6.2 and HuG6.3 (Thr73Lys mutant) to the biotinylated D80 scFv-Fc.

FIG. 8 shows binding of endogenous anti-H5 antibodies to H5 bycompetition ELISA. 3 ng of purified biotinylated-F10 (Bio-F10)antibodies were mixed with each serum sample at various dilutions andadded to H5 (H5-VN04)-coated plates, washed, and followed byHRP-streptavidin incubation to detect biotinylated-F10 bound to H5. Mostof serum samples, except No. 8, show ability to inhibit binding ofBio-F10 to H5, indicating the presence of endogenous anti-H5 antibodiesin serum samples from healthy individuals.

FIG. 9 shows binding of endogenous anti-H5 antibodies to H5 bycompetition ELISA. Purified biotinylated-F10 (Bio-F10) antibodies weremixed with serum samples obtained before or after H5N1 vaccination fromhealthy individuals. The mixture was added to H5 (H5-VN04)-coatedplates, washed, and followed by HRP-streptavidin incubation to detectbiotinylated-F10 bound to H5. Most of pre-vaccinated serum samples showability to inhibit binding of Bio-F10 to H5, indicating the presence ofendogenous anti-H5 antibodies in healthy individuals. H5N1 vaccinationboosts the production of anti-H5 antibodies in all of the individuals.The boosting effect is stronger in the individuals with lower amount ofendogenous anti-H5 antibodies.

FIG. 10 shows in vitro binding of acid-eluted and biotinylated-F10(Bio-F10)-eluted anti-H5 fractions from intravenous immunoglobulin(IVIG) samples. Purified F10 antibodies, acid-eluted, andbiotinylated-F10 (Bio-F10)-eluted anti-H5 fractions at variousconcentrations were evaluated for binding to various HA coated on anELISA plate. (A) Both acid-eluted and Bio-F10-eluted fractions recognizeH1-NY18 as control F10 antibodies do. (B) Both acid-eluted andBio-F10-eluted fractions recognize H5-VN04 as control F10 antibodies do.(C) Only control H3M3 and acid-eluted fraction recognize H3-BR07, butnot Bio-F10-eluted fraction. (D) Unlike the control antibody anti-H7,acid-eluted fraction only binds to H7-NL219 at high concentration, andBio-F10-eluted fraction does not recognize H7-NL219.

FIG. 11 shows FACS analysis of various anti-H5 eluate fractions bindingto H1, H2, H5 (Cluster H1a); and H8 (Cluster H9). 293T cells weretransiently transfected with different HA-expressing plasmids, andantibody binding to the cells was analyzed by FACS. H5-specific antibody80R is the negative control and F10 antibody with broader specificity isthe positive control. Both acid-eluted and Bio-F10 anti-H5 fraction canbind to H1, H2 and H5. They do not show much difference in binding toH1, H2 and H5. Complete viral strain designations are: H1-SC1918(A/South Carolina/1/1918 (H1N1)); H2-JP57 (A/Japan/305/57 (H2N2));H5-TH04 (A/Thailand/2-SP-33/2004(H5N1)); H8-ON68(A/Turkey/Ontario/6118/68).

FIG. 12 shows in vitro neutralization of various anti-H5 eluatefractions. Purified F10 antibodies, acid-eluted, and biotinylated-F10(Bio-F10)-eluted anti-H5 fractions at various concentrations wereevaluated for neutralizing activity against individual influenza virus.H5-specific antibody 80R is the negative control and F10 antibody withbroader specificity is the positive control. (A) Both acid-eluted andBio-F10-eluted fractions can neutralize H1-SC1918. (B) Neitheracid-eluted nor Bio-F10-eluted fractions can neutralize H2-JP57. (C)Both acid-eluted and Bio-F10-eluted fractions can neutralize H5-TH04.(D) Both acid-eluted and Bio-F10-eluted fractions can neutralize H6-NY98[H6-NY98 (A/Chicken/New York/14677-13/1998 (H6N2))].

FIG. 13 shows main germline nucleotide codons that distinguish thedifferent variants of the VH1-69 gene. Among 13 alleles, alleles *01,*03, *05, *06, *12, and *13 are 51p1 related.

FIG. 14 shows the distribution of antibodies that were isolated by usingG6 to pan against the Mehta I/II non-immune library. (A) 84% of theantibodies that bound to G6 were IGHV1-69 and they are primarilycomprised of *01 and *05 alleles which encode the critical Phe55 (FIG.6) that inserts into the hydrophobic pocket on the HA stem (Sui, NSMB'09). This data suggests that this anti-idiotype only recognizes the51p1 alleles but not the hv1263 alleles. (B) The random assortment of VLchains indicates that the G6 idiotype is only expressed on VH but notVL.

FIG. 15 shows the number of somatic mutations of the VH1-69 encodedantibodies that bound to G6.

FIG. 16 shows the frequency plot of the different amino acids that werefound at each of the positions of the VH1-69 encoded Abs that bound toG6. The frame with solid edges indicates complementarity determiningregion 1 (CDR1) and the frame with dashed edges indicates CDR2. Notethere is much less variability of critical amino acids that areimportant contact amino acids for HA binding such as the GGT in HCDR1and Phe55 in CDR2.

FIG. 17 shows predicated Activation-Induced Deaminase (AID) WRCY motifson VH1-69. The frame with solid edges indicates complementaritydetermining region 1 (CDR1) and the frame with dashed edges indicatesCDR2. The data shows that there is a paucity of WRCY motifs in the veryregions that are needed to mutate that permit rotation of Phe55 toinsert into the hydrophobic pocket (shown in the figures below).

FIG. 18 shows in vitro binding of anti-H5 antibodies. Six anti-H5 BnAbswere evaluated for binding to anti-idiotypic G6 mouse mAb. Only D8 showspositive binding to G6.

FIG. 19 shows in vitro binding of VH1-69/F10 to G6. (A) The cartoonillustrates the main domains of F10 and the domains of chimericconstruct VH1-69/F10. (B) VH1-69/F10 can bind to G6, but F10 cannot.This data confirms that the G6 idiotype is located in the Vh segment.

FIG. 20 shows schemes of different chimeric F10 constructs and theirbinding abilities to H5. Replacement of Pro with Ala outside the bindingdomain increases binding to H5 (+++++). However, the binding iscompletely lost when CDR2 is replaced with its germline form. Theintroduction of Ser back to the sequence recovers 90% of the binding.Replacement of framework regions (FR) with germline form also diminishesthe binding. Restoring several key binding residues on the germlinebackground rescues their binding abilities.

DETAILED DESCRIPTION

The present invention provides humanized monoclonal antibodies specificagainst human immunoglobulin heavy chain variable region germline geneVH1-69. In particular, the invention provides a humanized anti-humanVH1-69 idiotype antibody G6 (referred to herein as huG6). Morespecifically, the invention provides a method of augmenting an immuneresponse to an antigen by focusing an immune response to a humanvariable region germline gene in combination with antigenic stimulation.

Specifically, this invention is based upon a previous work in which highaffinity, cross-subtype, broadly-neutralizing human anti-hemagglutininmAbs were identified. (See, WO 2009/086514 and WO 2011/027818, thecontents of which are hereby incorporated by reference in theirentireties.) A human antibody phage display library and H5 hemagglutinin(HA) ectodomain was used to select ten neutralizing mAbs (nAbs) with aremarkably broad range among Group 1 influenza viruses, including theH5N1 “bird flu” and the H1N1 “Spanish flu” and “Swine flu” strains.These nAbs inhibit the post-attachment fusion process by recognizing anovel and highly conserved neutralizing epitope (referred to herein asthe F10 epitope) within the stem region at a point where key elements ofthe conformational change—the fusion peptide and the exposed surface ofhelix αA—are brought into close apposition.

Remarkably, these isolated nAbs utilizes the same VH germline gene,IGHV1-69*01, and encodes a CDR3 loop containing a tyrosine at anequivalent position to Y102, from a non-immune library. This indicatedthat broad anti-HA cross-immunity pre-exists in the H5-naive population.The recurrent use of this germline VH segment, the commonality of theCDR3 tyrosine introduced through N insertion and/or germline D geneassembly, and the promiscuous use of VL genes by the discovered nAbsdiscovered indicate that the precursor frequency of rearranged VHsegments that could recognize this epitope is significant. Thisindicates that with suitable exposure to the F10 epitope, thesebroad-spectrum nAbs can be readily induced in vivo.

The hemagglutinin of the influenza virus has to functions that areessential for the initiation of the influenza virus infection andinvolve two structurally distinct regions, the globular head and thestem region. The globular head region contains the main antigenicdeterminants in which the antigenic mutations arise. The stem regionhowever is conserved. Thus, finding of these broadly-neutralizing humananti-hemagglutinin mAbs suggest that with proper antigenic stimulation,humans are capable of eliciting broad neutralizing anti-influenzaresponse.

Anti-idiotype antibodies (termed Ab2) are antibodies directed againstthe variable region (antigen-binding site) of another antibody (Ab1),the idiotype. In turn, immunization with Ab2 antibodies can induceantibodies with specificities similar to the original antibodies. In thepresent invention it is proposed to immunize with an anti-idiotypicantibody that is specific for immunoglobulin variable region germlinegene to clonotypically stimulate a germline gene immune response. Thiswill in effect prime the immune system by activating germline genespecific B-cells.

Accordingly, in one aspect the invention provides a method or augmentingan immune response in a subject to an antigen by administering to thesubject an anti-immunoglobulin variable region germline gene idiotypeantibody and an immunogen capable of inducing an immune reaction to theantigen. For example, the variable region germline gene is VH1-69.

In another aspect the present invention provides a humanized monoclonalantibody that specifically binds human immunoglobulin heavy chainvariable region protein encode by germline gene VH1-69. The huG6antibody is monovalent or bivalent and comprises a single or doublechain. Functionally, the binding affinity of the huG6 antibody is lessthan 250 nM, less than 100 nM, less than 10 nM, less than 1 nM, or lessthan 100 pM. Preferably, the binding affinity is within a range of 100pM-1 nM. The sequence of the antibody is engineered from and thus, maycomprises one or more antigen-binding regions of murine antibody G6. ThehuG6 antibody binds the same epitope as murine Mab G6. Furthermore, theantibody comprises an immungen (i.e., antigen) including, but notlimited to, the hemagglutinin (HA) protein of an influenza virus orfragment thereof. For example, the immunogen comprises the stem regionof the hemagglutinin (HA) protein of an influenza virus.

The murine G6 (muG6) single-chain antibody (1567) was constructed fromthe hybridoma cell clone G6. Upon cloning, it was discovered that the G6hybridoma encoded three different variable light chain genes. All thelight chains were Vk. The murine G6 VH and VL nucleic acid and aminoacid sequences are as follows:

muG6 VH nucleotide sequence: (SEQ ID NO: 13)CAGGTCCAGCTGCAGCAGTCTGGGACTGTGCTCGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGTTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTGTTTCTCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGTCACATCCACCAGCACTGCCTACATGGAGTTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGAAGTCGATATGGTAACAATGCTTTGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAmuG6 VH amino acid sequence: (SEQ ID NO: 14)QVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAVSPGNSDTSYNQKFKGKATLTAVTSTSTAYMEFSSLTNEDSAVYYCTRSRYGNNALDYWGQGTTVTVSSmuG6-19 V_(L) nucleotide sequence: (SEQ ID NO: 15)GACATCGAGCTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAagGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA muG6-30 V_(L) nucleotide sequence:(SEQ ID NO: 16)GACATCGAGCTCACTCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAGAGCCAGCGCAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAACCTATTACTGTCAGCACATTAagGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAA muG6-39 V_(L) nucleotide sequence:(SEQ ID NO: 17)GACATCGAGCTCACTCAGTCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAACATCACTTGCCGTGCAAGTCAGGGCATTAGCAGTAATATAGTGTGGTTGCAGCAGAAACCAGGGAAGTCATTTAAGGGCCTGATCTATCATGGGACCAATTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAGGATTTTGCAGACTATTACTGTGTACAGTATTCTCAGTTTCCTCCCACGTTCGGCTCGGGGACCAAGCTGGAGCTGAAA muG6 V_(L) amino acid sequence: (SEQ ID NO: 18)DIELTQSPSSMSVSLGDTVNITCRASQGISSNIVWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYSQFPPTFGSGTKLELK

The heavy chain CDRs of the huG6 antibody have the following sequences:CDRH1: GYTFTSYW (SEQ ID NO: 5); CDRH2: VSPGNSDT (SEQ ID NO: 6); andCDRH3: TRSRYGNNALDY (SEQ ID NO: 7). The light chain CDRs of the huG6antibody have the following sequences: CDRL1: QGISSNIVW (SEQ ID NO: 8);CDRL2: HGT (SEQ ID NO: 9); and CDRL3: VQYSQFPPT (SEQ ID NO: 10). Thenucleotide VH and VL sequences were optimized for mammalian codon usage.

huG6.2 V_(H ) nucleotide sequence: (SEQ ID NO: 1)CAGGTCCAGCTCGTCCAGTCCGGCGCTGAAGTGGTGAAACCCGGGGCATCCGTCAAAGTCTCTTGTAAGGCTAGTGGCTACACCTTCACATCCTACTGGATGCATTGGGTGAAACAGGCACCTGGCCAGGGACTCGAATGGATCGGAGCCGTGTCTCCTGGAAATTCCGACACCTCCTACAACGAAAAATTCAAGGGCAAGGCAACCCTCACTGTGGATACTAGTGCTTCTACCGCCTACATGGAACTCTCATCTCTCCGCTCTGAGGACACTGCCGTCTACTACTGTACTCGGTCACGATACGGGAACAACGCTCTCGATTACTGGGGACAGGGCACACTGGTCACTGTCTCThuG6.2 V_(H ) amino acid sequence: (SEQ ID NO: 2)

huG6.2 V_(L ) and huG6.3 V_(L ) nucleotide sequence: (SEQ ID NO: 3)GATATTCAGCTCACACAGAGCCCATCTTCTCTGTCTGCTTCTGTGGGCGATCGAGTGACAATCACTTGTCGGGCTAGTCAGGGCATTTCTAGCAACATTGTGTGGCTCCAGCAGAAACCTGGCAAAGCCCCAAAAGGCCTCATCTACCACGGAACCAACCTGGAATCTGGCGTGCCATCTCGGTTTAGTGGATCTGGATCCGGGACCGATTACACACTCACCATCTCTTCACTGGAACCTGAGGATTTCGCCACCTACTACTGTGTCCAGTACTCCCAGTTTCCACCCACTTTTGGACAGGGAACCAAACTCGAGATCAAAhuG6.2 V_(L ) and huG6.3 V_(L ) amino acid sequence: (SEQ ID NO: 4)DIQLTQSPSSLSASVGDRVTITCRASQGISSNIVWLQQKPGKAPKGLIYHGTNLESGVPSRFSGSGSGTDYTLTISSLEPEDFATYYCVQYSQFPPTFGQGTKLEIK huG6.3 V_(H ) nucleotide sequence:(SEQ ID NO: 11)CAGGTCCAGCTCGTCCAGTCCGGCGCTGAAGTGGTGAAACCCGGGGCATCCGTCAAAGTCTCTTGTAAGGCTAGTGGCTACACCTTCACATCCTACTGGATGCATTGGGTGAAACAGGCACCTGGCCAGGGACTCGAATGGATCGGAGCCGTGTCTCCTGGAAATTCCGACACCTCCTACAACGAAAAATTCAAGGGCAAGGCAACCCTCACTGTGGACAAATCTGCCTCTACCGCCTACATGGAACTCTCATCTCTCCGCTCTGAGGATACTGCTGTGTACTACTGTACCCGGTCACGATACGGCAATAACGCCCTCGATTACTGGGGGCAGGGAACTCTGGTCACTGTGTCThuG6.3 V_(H ) amino acid sequence: (SEQ ID NO: 12)

Antibodies

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” is meant that the antibody reacts with one or moreantigenic determinants of the desired antigen and does not react withother polypeptides. Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and F_(ab) expressionlibraries.

A single chain Fv (“scFv”) polypeptide molecule is a covalently linkedV_(H)::V_(L) heterodimer, which can be expressed from a gene fusionincluding V_(H)- and V_(L)-encoding genes linked by a peptide-encodinglinker. (See Huston et al. (1988) Proc Nat Acad Sci USA85(16):5879-5883). A number of methods have been described to discernchemical structures for converting the naturally aggregated, butchemically separated, light and heavy polypeptide chains from anantibody V region into an scFv molecule, which will fold into a threedimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405;and 4,946,778.

Very large naïve human scFv libraries have been and can be created tooffer a large source of rearranged antibody genes against a plethora oftarget molecules. Smaller libraries can be constructed from individualswith infectious diseases in order to isolate disease-specificantibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43(1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)).

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain. The term“antigen-binding site” or “binding portion” refers to the part of theimmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”Specifically, the CDRs of the antibody heavy chains are referred to asCDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of theantibody light chains are referred to as CDRL1, CDRL2 and CDRL3,respectively.

An idiotype is the genetically determined variation of intramolecularstructures in the variable regions of immunoglobulins. T. However,idiotype variation involves the amino acid sequence and proteinstructure (so-called determinants) especially in the area of theantigen-binding site, also referred to as the “idiotope”. The term“idiotype” designates the complete set of determinants of a variableregion of an antibody molecule.

An anti-idiotype antibody may be generated with a process that uses apurified human monoclonal antibody or a human hybridoma cell line thatexpresses a human monoclonal antibody. For example a process forgeneration of an anti-idiotype antibody may involve culturing a humanhybridoma cell line that secretes a human monoclonal antibody into itssupernatant and purifying this antibody, for example, using affinitychromatography, ion exchange chromatography, gel filtration, or acombination thereof. This purified human monoclonal antibody may then beused to immunize a non-human mammal, such as a mouse or a rat, by meansof, for instance, an intraperitoneal injection or in vitro directly onisolated B lymphocytes. B lymphocytes may then be isolated from thenon-human mammal sacrificed up to four days after the last immunization,and the isolated B lymphocytes may be brought into contact with myelomacells of same species (e.g., mouse or rat) under conditions that lead tofusion of the myeloma cells with the B lymphocytes to generate anon-human hybridoma cell. These non-human hybridoma cells can then becultured and tested (e.g., using ELISA) for expression of idiotype Igantibodies, e.g., IgM, IgA, or IgG antibodies, after, for example, threeweeks of culturing. These Ig antibodies can be tested for specificbinding to the human hybridoma cells and to various antibodies,including the human monoclonal antibody used to immunize the non-humanmammal.

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. For example, antibodies maybe raised against N-terminal or C-terminal peptides of a polypeptide.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to an epitope when the equilibrium bindingconstant (K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably ≦10 nM,and most preferably ≦100 pM to about 1 pM, as measured by assays such asradioligand binding assays or similar assays known to those skilled inthe art.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the inventionby ascertaining whether the former prevents the latter from binding to ahuman immunoglobulin variable region polypeptide. If the humanmonoclonal antibody being tested competes with the human monoclonalantibody of the invention, as shown by a decrease in binding by thehuman monoclonal antibody of the invention, then it is likely that thetwo monoclonal antibodies bind to the same, or to a closely related,epitope.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference).

Antibodies can be purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The term “monoclonal antibody” or “MAb” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanizedantibodies”, “human antibodies”, or “fully human antibodies” herein.Human monoclonal antibodies can be prepared by using trioma technique;the human B-cell hybridoma technique (see Kozbor, et al., 1983 ImmunolToday 4: 72); and the EBV hybridoma technique to produce humanmonoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized and may be produced by using human hybridomas(see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv (scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci. USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively,an antibody can be engineered that has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. (See Stevenson etal., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987)). Preferred linkers aredescribed in the literature. (See, for example, Ramakrishnan, S. et al.,Cancer Res. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Methods of Treatment

The antibodies can be used to prevent, diagnose, or treat medicaldisorders in a subject, especially in humans. The invention provides forboth prophylactic and therapeutic methods of treating a subject at riskof (or susceptible to) or having an infection. The infection is viral,bacterial, fungal, or parasitic.

The present invention provides methods of augmenting the immune responseof a subject to an antigen comprising administering to said subject anantibody or fragment thereof that recognizes a human heavy chainvariable region germline geneVH1-69 and an immunogen capable of inducingan immune reaction to said antigen. In some aspects the antibody ishuG6.2. In another aspect, the antibody is huG6.3. In some aspect theantibody includes a heavy chain variable region (SEQ ID NOS: 2 or 12),encoded by the nucleic acid sequence SEQ ID NOS: 1 or 11, and a lightchain variable region (SEQ ID NO: 4) encoded by the nucleic acidsequence SEQ ID NO: 3. The heavy chain CDRs of the antibody have thefollowing sequences: SEQ ID NOS: 5, 6 and 7. The light chain CDRs of theantibody have the following sequences: SEQ ID NOS: 8, 9 and 10.Preferably the three heavy chain CDRs include an amino acid sequence ofat least 90%, 92%, 95%, 97%, 98%, 99%, or more identical to the aminoacid sequence of SEQ ID NOS: 5, 6 and 7 and a light chain with threeCDRs that include an amino acid sequence of at least 90%, 92%, 95%, 97%,98%, 99%, or more identical to the amino acid sequence of SEQ ID NOS: 8,9 and 10.

In some aspects, the immunogen is covalently linked to the antibody. Theantigen could be a virus, a bacterium, a fungus, or a parasite. Theimmunogen is of viral (e.g. influenza, HIV), bacterial, fungal origin.For example, the immunogen is the hemagglutinin (HA) protein of aninfluenza virus or fragment thereof. Preferably, the immunogen comprisesthe stem region of the hemagglutinin (HA) protein of an influenza virus.The antibody of this invention can be administered prior to,concurrently with, or subsequent to the administration of the immunogen.

Included in the invention are methods of increasing or enhancing animmune response to an antigen. An immune response is increased orenhanced by administering to the subject an anti-immunoglobulin variableregion germline gene idiotype antibody and an immunogen. In a preferredembodiment, the antibody includes a heavy chain variable region (SEQ IDNOS: 2 or 12), encoded by the nucleic acid sequence SEQ ID NOS: 1 or 11,and a light chain variable region (SEQ ID NO: 4) encoded by the nucleicacid sequence SEQ ID NO: 3. The heavy chain CDRs of the antibody havethe following sequences: SEQ ID NOS: 5, 6 and 7. The light chain CDRs ofthe antibody have the following sequences: SEQ ID NOS: 8, 9 and 10.Preferably the three heavy chain CDRs include an amino acid sequence ofat least 90%, 92%, 95%, 97%, 98%, 99%, or more identical to the aminoacid sequence of SEQ ID NOS: 5, 6 and 7 and a light chain with threeCDRs that include an amino acid sequence of at least 90%, 92%, 95%, 97%,98%, 99%, or more identical to the amino acid sequence of SEQ ID NOS: 8,9 and 10.

In some embodiments the germline gene is VH1-69. In some aspects theantibody is huG6.2. In another aspect, the antibody is huG6.3. Theantigen could be a virus, a bacterium, a fungus, or a parasite. Theimmunogen is of viral (e.g. influenza, HIV), bacterial, fungal origin.For example, the immunogen is the hemagglutinin (HA) protein of aninfluenza virus or fragment thereof. Preferably, the immunogen comprisesthe stem region of the hemagglutinin (HA) protein of an influenza virus.The antibody of this invention can be administered prior to,concurrently with, or subsequent to the administration of the immunogen.

Pharmaceutical Compositions

The antibodies or agents of the invention (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the antibody oragent and a pharmaceutically acceptable carrier. As used herein, theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, and foreign patents, foreign patentapplications referred to in this specification, are incorporated hereinby reference in their entirety.

All publications cited in the specification are indicative of the levelof skill of those skilled in the art to which this invention pertains.All these publications are herein incorporated by reference in theirentirety to the same extent as if each individual publication werespecifically and individually indicated to be incorporated by reference.

EXAMPLES Example 1 Humanization of Mouse G6 Antibody

1. Homology Model of Mouse G6 Antibody Using Web Antibody Modeling(WAM).

Mouse G6 variable heavy (VH) and variable light (VL) chain amino acidsequence were submitted to Web based Antibody Modeling program (WAM)¹for generating the homology model of mouse G6 antibody. The WAM programtakes into account various parameters which govern differentconformations of the antibodies. In antibodies, usually the residues inthe framework region are found to be mostly conserved whereas theresidues which form the complementary determining regions (CDRs) are themost variable. As such homology based approach is used, five of the sixCDRs (except Heavy chain CDR3) can be categorized into classicalcanonical structure classes. In addition, members of the same canonicalclasses have very similar loop conformation. This is determined by thelength of the loop, presence of certain conserved key residues not onlyin framework regions but also in the CDRs.

Briefly WAM carried out different computational parameters in itsalgorithm to create a homology model of mouse G6 antibody format fromits amino acid sequence using the following criteria:

a) The framework residues of a mouse G6 antibody which not only includesbackbone residues but also side chain residues together with canonicalloop backbone residues were built using the most known (X-ray and NMR)homology structures.

b) Using uniform conformational sampling with iterative algorithmCONGEN, predicted the conformation of side chains in the loops as toobtain the most energetically minimized conformation².

c) The non-canonical loop regions were modeled through a series ofconformations obtained from protein data bank database search (PDB) orusing different conformational/structure based database search for aloop conformation.

d) The different conformations generated using the previous step wasfurther energy minimized using Eureka, which is a solvent-modifiedversion of Valence Force field (VFF)³). In addition, root mean squaredeviation (R.M.S.D) screen which compares similarity in the r.m.s.d tothe known heavy chain CDR3 (H3) structures containing same length as themodeling candidate was also used.

The final model of mouse G6 antibody was selected from the first fivelowest energy conformations. The algorithm compared the torsion anglesbetween the model candidate and the original set of loops from a knownstructure in protein data bank. The final model with conformationclosest to the set of torsion angles was selected and displayed in FIG.1.

2. Humanization of Mouse G6 and Generation of the 1^(st) Version ofHumanized G6 (huG6.1) on the Basis of G6's Closest Human GermlineSequence and Surface Accessibility of Residues in Frameworks of Mouse G6Homology Model

The mouse G6 homology model generated from WAM was used as a template toidentify residues which are surface accessible (solvent exposed) asvisualized through DeepView program, using a 30% threshold limit⁴⁻⁵.Subsequently mouse G6 variable heavy chain (VH) and light chain (VL)residues were searched using IGBLAST (www.ncbi.nlm.nih.gov/igblast/)against the human IgG germline database. To humanize mouse G6, weexchanged the surface accessible residues in frameworks of both VH andVL manually to those found in the selected human germline sequence, thusgenerating humanized G6 version 1 (hG6.1). Sequence alignment is shownbelow (Total 14 amino acids changes in VH, and 15 in VL).

VH

VL

Next, human G6.1 single chain variable region (scFv) was de novosynthesized by Genewiz and the gene was codon-optimized for mammaliancell expression. The scFv-Fc fragment of huG6.1 was constructed bysubcloning the synthesized scFv into a Fc expression vectorpcDNA3.1-Hinge which contains the hinge, CH2 and CH3 domains of humanIgG1 but lacks CH1 domain. Human G6.1 scFv-Fc was expressed in 293Tcells (ATCC) by transient transfection (Lipofectamine, Invitrogen) andpurified by protein A sepharose affinity chromatography. To test theantigen (herein is D80-IgG1 that uses the 51p1 form of VH1-69) bindingactivity of huG6.1 scFv-Fc and compare it with mouse G6, we biotinylatedhuG6.1 and mouse G6 with a commercial biotinylation kit (Pierce) and didELISA analysis. Briefly, D80-IgG was coated on to a 96 well Maxisorpplate at 2 μg/ml, overnight at 4° C. Unbound protein was washed awaywith PBS and the plate was blocked with 2% milk/PBS for 1 hour at 25° C.Diluted biotinylated HuG6.1 scFv-Fc and Mouse G6 scFv-Fc was added tothe wells and incubated at 25° C. for 1 hour. Plates were washed withPBST (0.05% Tween/PBS). Streptavidin-HRP was added, incubated at 25° C.for 30 minutes. Plates were washed again and developed with TMB solutionand 5 minutes later reaction was stopped with addition of stop reagent.Plate was read at OD450. As shown in FIG. 2, huG6.1 lost antigen bindingactivity significantly as compared to mouse G6 (FIG. 2)

3. Energy Minimization of huG6.1 to Improve the Humanization.

Next, we applied GROMOS force field energy minimization parameter tohomology model huG6.1 using DeepView program⁶. The final model wasvisualized with DeepView and PyMOL programs as shown in FIG. 3a .Examination of this energy minimized homology model of Human G6.1resulted in the identification of certain residues which had distortedgeometry or steric clashes between different residues in framework aswell as complementary determining region residues (CDRs) as shown inFIG. 3 b.

4. Generate huG6.2 and G6.3 Antibody in Order to Ameliorate DistortedGeometry and Steric Clashes Using PyMOL Program.

Closer examination in DeepView program displayed certain residues withhigh entropy in their side chain rotamers. These anomalies revealedresidues with distorted geometry and steric clashes between residueswithin framework regions as well as between framework and CDR residues.Further inspection in PyMOL led to the following observations:

i) Lysine⁷³ (Lys⁷³) (kabat numbering being followed throughout) in theVH steric clashed with Glycine⁵³ (Gly⁵³) in CDR2 of VH (FIG. 4a ).Because CDR residues should not be changed, the Lys⁷³ (in huG6.3) waschanged back to Thr of mouse G6 to solve the steric clash.ii) Methionine⁴ (Met⁴) had a steric clash with the conserved cysteine⁸⁸(Cys⁸⁸) residue which is positioned right before CDR3 of the light chainvariable region as shown in left panel in FIG. 4b . Sequence alignmentrevealed that Cys⁸⁸ is highly conserved residue and Met⁴ is not,therefore Met⁴ can be backmutated to Leucine residue as found in mouseG6. This back mutation solved the steric clash between the residues asshown in the right panel in FIG. 4 b.iii) Tyrosine³⁶ (Tyr³⁶) in the light chain framework 2 also had stericclash with Leucine^(100B) (Leu^(100B)) in the CDR3 of heavy chain theleft panel FIG. 4c . As such Tyr³⁶ can be back mutated back to Leu³⁶(mouse residue) which solves the steric clash between the residues asshown in right panel in FIG. 4 c.iv) Glutamine⁷⁹ (Gln⁷⁹) residue in framework 3 (light chain) region hadsteric clash with Arginine⁶¹ (Arg⁶¹) of the same framework left panel inFIG. 4d . Sequence alignment indicated that Arg⁶¹ is mostly conservedamong different homologous sequences whereas Gln⁷⁹ was not conserved. Assuch Gln⁷⁹ can be back mutated to Glutamate⁷⁹ in the light chain whichfixs the steric clash as shown in right panel in FIG. 4 d.

In summary, we identified four residues which can be changed back tomouse residues: one residue in VH (Thr73) and three residues in VL(Leu4, Leu36, Glu79). Based on this structural analysis result, we madehumanized G6 version 2 (huG6.2) and version 3 (huG6.3). In huG6.2, thefour residues were changed back to mouse residues. In huG6.3, only the 3amino acids in VL were changed back to mouse residues, in another words,there is only one amino acid difference between huG6.2 and huG6.3 whichis mouse residue Thr73 in VH of huG6.2, while human Lys73 in VH ofhuG6.3. The amino acid sequence differences among the huG6.1, huG6.2,huG6.3 and mouse G6 genes are shown below, highlighted in pink are thefour residues.

Multiple Sequence Alignment of huG6.1, huG6.2, huG6.3 and Mouse G6.

VH

VL

5. Comparing Antigen Binding Activity of huG6.2 and Mouse G6.

Human G6.2 scFv-Fc was synthesized, expressed, purified and biotinylatedusing the same method as described above for huG6.1. ELISA was performedto compare huG6.2 and mouse G6. As shown in FIG. 5, huG6.2 has similarbinding activity to D80 as the mouse G6. Also it is consistent with thedata shown in FIG. 2, huG6.1 only binds very weakly to D80. Thisdemonstrated that the four residue changes from huG6.1 back to mouseresidues—huG6.2 indeed play critical important role in restoring thebinding activity of humanized G6.

We also compared huG6.2 scFv-Fc and mG6 scFv-Fc using Octet Redinstrument (ForteBio, Menlo Park, Calif., USA) that utilize Bio-LayerInterferometry (BLI), a label-free technology to measure protein-proteininteraction. For this assay, Antigen for G6, D80-scFv-Fc, wasbiotinylated and coated on streptavidin (SA) biosensor tips (ForteBio,Menlo Park, Calif., USA). The assay was performed at 30° C. in 1×kinetics assay buffer (0.1 mg/ml BSA, 0.002% Tween-20, PBS). Sampleswere agitated at 1000 rpm. Prior to experimental run, the SA sensorswere humidified in PBS for 15 minutes. SA sensor tips were loaded with20 μg/ml of biotinylated D80 scFv-Fc for 900 secs which typicallyresulted in capture levels of 3.0-3.5 nM. G6 antibodies were prepared in100 nM concentration. Association and dissociation rates were monitoredfor 300 secs. Data was processed and analyzed using the Octet dataanalysis software (ForteBio). As shown in FIG. 6. HuG6.2 (red) has aslower association and dissociation rates than that of mouse G6 (blue).The affinity for both are within a range of 100 pM-1 nM.

6. Kinetic Analysis of the Binding Activity of HuG6.2 scFv-Fc and HuG6.3(Thr73Lys mutant) scFv-Fc to Biotinylated D80.

The Bio-Layer Interferometry was performed as described above. Brieflythe only change was four different concentrations of Human G6.2 andHumanG6.3 scFv-Fcs were used (100 nM, 10 nM, 1 nM and 0 nM). Theassociation and dissociation rates were monitored for 300 secs and 4000secs respectively. The results showed that HuG6.3 had faster associationrates (on) and slower dissociation rates (off) as compared to Human G6.2as shown in FIG. 7. This suggested that residue Lys⁷³ in the framework 3region of the VH of Hu6.3 is important in binding to D80.

In summary, we have made humanized G6.2 and G6.3 that have similar orbetter binding activity/kinetics as compared to mouse G6. The estimatedaffinity for them is within range of 100 pM-1 nM.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

REFERENCES

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What is claimed is:
 1. An isolated humanized monoclonal antibody,wherein said antibody has a humanized heavy chain with three CDRscomprising an amino acid sequence of SEQ ID NO: 5 (GYTFTSYW); SEQ ID NO:6 (VSPGNSDT); and SEQ ID NO: 7 (TRSRYGNNALDY); and a humanized lightchain with three CDRs comprising an amino acid sequence of QGISSNIVW SEQID NO: 8 (QGISSNIVW); SEQ ID NO: 9 (HGT); and SEQ ID NO: 10 (VQYSQFPPT),wherein said antibody binds human immunoglobulin heavy chain variableregion protein encoded by germline gene VH1-69.
 2. A compositioncomprising an isolated humanized monoclonal antibody, wherein saidantibody has a humanized heavy chain with three CDRs comprising an aminoacid sequence of SEQ ID NO: 5 (GYTFTSYW); SEQ ID NO: 6 (VSPGNSDT); andSEQ ID NO: 7 (TRSRYGNNALDY); and a humanized light chain with three CDRscomprising an amino acid sequence of QGISSNIVW SEQ ID NO: 8 (QGISSNIVW);SEQ ID NO: 9 (HGT); and SEQ ID NO: 10 (VQYSQFPPT); wherein said antibodybinds human immunoglobulin heavy chain variable region protein encodedby germline gene VH1-69; the composition further comprising an antigen;and wherein said antigen comprises the stem region of the hemagglutinin(HA) protein of an influenza virus.
 3. The composition of claim 2,wherein the antibody is a single chain antibody.
 4. The composition ofclaim 2, wherein the antigen is covalently linked to said antibody.